properties. Other examples include the reorganization of cholesterol-rich bilayers due to the addition of polyunsaturated fatty acids (Masonet al., 2016),α-synuclein peptides (Pirc and Ulrih, 2015), and inter-actions of amyloidβ-peptide with lipid bilayers (Suzuki and Miura,2015), interactions of dendrimers with artificial bilayers and mono-layers (Melikishvili et al., 2016), or toxin-induced pore formation(García-Linares et al., 2016). DPH is also frequently used to study theeffect of drugs and drug-like small molecules on lipid bilayer properties(Alves et al., 2017; Balducci et al., 2018;De Athayde et al., 2016; dosSantos et al., 2017; Giudice et al., 2016; Miguel et al., 2018; Neveset al., 2016,2015; Vaňousová et al., 2018; Yun and Lee, 2017; Yunet al., 2015). The use of DPH is not limited to model bilayer studies,intact biological membranes are also investigated using the DPH probe(Brejchová et al., 2015;Cerecedo et al., 2016; Wang et al., 2018,2016;Yun et al., 2018).The effects of DPH on lipid bilayers has been investigated usingexperimental and MD simulations methods. Repáková et al. used dif-ferential scanning calorimetry,2H NMR measurements, and atomisticMD simulations to study the behavior of DPH embedded in the satu-rated (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC) bilayer inthe liquid-crystalline phase (Repáková et al., 2005, 2004). They foundthat the probe slightly increases the lipid acyl tail order in the DPPCbilayer and has a weak effect on the DPPC phase transition. In the caseof the mixed DPPC/Chol (5 and 20 mol %) bilayer, it was shown by MDsimulations that DPH affects the membrane properties, but the DPH-induced perturbations are local and depend on the concentration ofChol in the membrane (Fraňová et al., 2010). Recently, MD simulationsof DPH behavior in the 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocho-line (POPC) and POPC/Chol bilayers were performed (do Canto et al.,2016). These studies showed a weak ordering effect of the probe on thePOPC bilayer, with the ordering being stronger at the end of the acyltails. The effect of DPH on the POPC/Chol bilayer was even weaker;only the segments at the beginning of the acyl tail were slightly dis-ordered.In this study, we employed atomic-scale MD simulations combinedwith steady-statefluorescence anisotropy andfluorescence lifetimemeasurements andfluorescence recovery after photobleaching (FRAP)experiments to evaluate the usefulness of the DPH probe as a reporteron the properties of zwitterionic membranes doped with a hydrophobicdrug. We used itraconazole (ITZ) as a model hydrophobic drug. ITZ isan orally administered triazole antifungal agent frequently used to treatsevere mycotic infections in both regular and immunocompromisedpatients (Clissold and Grant, 1989). First, the effect of ITZ on theproperties of the POPC bilayer was examined. Next, we compared thebehavior of DPH in the pristine and ITZ-doped POPC bilayer. Our re-sults show that the location and orientation of DPH is strongly affectedby the drug molecules that adopt an orientation parallel to the mem-brane surface.2. Materials and methods2.1. MaterialsItraconazole (ITZ), synthetic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 2,2′-(1,4-phenylene)bis[5-phenyl-oxazole](POPOP), sucrose, and glucose were purchased from Sigma-Aldrich.1,6-Diphenyl-1,3,5-hexatriene (DPH, forfluorescence,≥97.5%) wasreceived from Fluka. 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanola-mine-N- (lissamine rhodamine B sulfonyl) (DPPE-Rh) was purchasedfrom Avanti Polar Lipids (Alabaster, AL). Low gelling temperatureagarose was purchased from Fisher Scientific (Waltham, MA). All sol-vents were obtained from Aldrich. Dimethylformamide (DMF) was ofspectroscopic grade. Milipore-quality water was used during the ex-periments.2.2. Preparation of liposomesPOPC/ITZ small unilamellar vesicles (SUVs) were prepared by so-nication as described previously (Kepczynski et al., 2010). Briefly, stocksolutions of POPC (34.6 mM) and ITZ (9.5 mM) were prepared inchloroform. Appropriate volumes of the stock solutions were combinedin a volumetricflask,and then the solvent was evaporated under astream of nitrogen to complete dryness. The dryfilm was hydrated with10 mM phosphate buffer pH 7.4. Thefinal concentration of POPC was2.5 mg/mL. The size of POPC/ITZ liposomes was in the range of30–200 nm (Dzieciuch-Rojek et al., 2017). Giant unilamellar vesicles(GUVs) were prepared by electroformation (Lira et al., 2014), see also(Dimova, 2019). A stock solution containing 3 mM of POPC and 0.5 mol% of DPPE-Rh was prepared in chloroform and mixed with appropriatevolumes of the ITZ stock solution. TenμL of the solution were spread ona pair of conductive glasses coated with indium tin oxide. The solventwas evaporated under a stream of nitrogen for 5 min. The glasses weresandwiched using a 1 mm Teflon spacer, forming a 1.5 ml chamber. A0.2 M sucrose solution was added to the chamber to hydrate the lipidfilms and the chamber was connected to a function generator. A 10 HzACfield of 1 V amplitude was applied, and the vesicles were allowed togrow for 1 h at room temperature in the dark. The vesicles were har-vested and dispersed in an isotonic glucose solution for subsequentimaging and analysis.2.3. Steady-statefluorescence anisotropy measurementsDPH was dissolved in DMF to form a stock solution. The POPC/ITZliposomes were stained with DPH in the dark for 1 h. Thefinal con-centration of DPH was 1.02 × 10−7M. Anisotropy measurements wereperformed as described previously (Kepczynski et al., 2011) using anSLM-AMINCO 8100 spectrofluorimeter working in the L-format andequipped with automatic polarizers. Samples were excited atλexc= 350 nm and thefluorescence intensity was monitored atλem= 428 nm. The steady state anisotropy (r) was calculated using thefollowing equation (Lakowicz, 2006):=−+rIGIIGI2vvvhvvvh(1)whereIis thefluorescence intensity,‘v’and‘h’subscripts denote thevertical and horizontal settings of the excitation and emission polar-izers, respectively.Gis an instrumental correction factor and was cal-culated individually for each sample according to the equation:=GIIhvhh(2)The apparent microviscosity was calculated using Eq.(3) (Pandeyand Mishra, 1999).=−ηrr ̄2.40.362(3)2.4. Fluorescence lifetimes of DPHAllfluorescence measurements were performed in 1.5 ml quartzcuvettes. The temperature of (293 ± 0.5) K was maintained using awater-circulating thermostat. Samples were equilibrated for 10 minbefore each measurement. Steady-statefluorescence spectra was col-lected using a Fluorolog-3 spectrofluorimeter (model FL3-11,JobinYvon Inc., Edison, NJ, USA). Fluorescence decays were recordedon a time-correlated single-photon counting (TCSPC) spectrometer,model 5000 U SPC, equipped with a 375 nm NanoLED 11 diode laserand a cooled Hamamatsu R3809U-50 microchannel plate photo-multiplier (IBH, Glasgow, UK). The excitation and emission wave-lengths (EX/EM = 375/466 nm) were chosen using monochromatorsand a 399 nm cut-offfilter was used to eliminate scattered light. TheC. Poojari, et al.Chemistry and Physics of Lipids 223 (2019) 1047842